The objective of this research is to characterize the cells and regulatory interactions responsible for an in vivo secondary humoral immune response in man. Pursuant to this objective we intend to in vivo booster immunize individuals with tetanus toxoid and functionally and numerically quantitate changes in the subpopulations of regulatory lymphocytes using in vitro techniques. Initially we will use limiting dilution analyses of B-lymphocytes and helper and suppressor T-lymphocytes to determine whether PWM stimulation of anti-tetanus antibody synthesis is polyclonal (i.e., polyspecific) or non-specific. Next, we will determine what changes in antigen specific regulatory T-lymphocyte subpopulations (both helper and suppressor) are responsible for the induced synthesis of anti-tetanus toxoid antibody in vitro following in vivo immunization. We will then examine the kinetics of appearance of subpopulations of tetanus toxoid reactive B-lymphocytes and helper and suppressor T-lymphocytes in the circulation following booster immunization and attempt to correlate in vitro cellular interactions among these subpopulations with the in vivo response. To determine whether the results observed in vitro are truly representatives of in vivo cellular interactions, we will compare by isoelectric focusing the clonality of anti-tetanus toxoid antibodies induced in vivo with those produced in vitro. Lastly we will define the role of specific portions of the tetanus toxoid molecule in the regulation of the immune response. This will be accomplished by employing fragments of tetanus toxoid molecule as probes for specific epitopes responsible for T-lymphocyte proliferation, B-cell recognition and T-lymphocyte suppression. Methods employed include mitogen and antigenic stimulation of peripheral blood lymphocytes follow by analysis of specific antibody and total immunoglobulin by either quantitative radioimmunoassays or plaque techniques.